RESUMO
Angiotensin (ANG) II is now recognized as a powerful direct controller of Na+ reabsorption in the proximal convoluted tubule, a property that predominantly reflects stimulation of the transepithelial NaHCO3 flux. Numerous studies have established that this effect of ANG II represents stimulation of the apical Na+/H+ exchanger, but a single microperfusion study has also suggested direct stimulation of the basolateral Na(+)-HCO3- cotransporter. We have carried out studies in basolateral membrane vesicles from rabbit renal cortex to examine directly whether ANG II exerts an independent effect on the Na(+)-HCO3- cotransporter. Preincubation of vesicles with ANG II (10(-11) to 10(-9) M) for 15 min enhanced the activity of the cotransporter, the greatest effect occurring at 10(-11) M (41 +/- 1.1%, P < 0.005). This stimulation reflected an increase in the maximal enzyme reaction velocity of the cotransporter but no change in the Michaelis constant for Na+. ANG II had no effect on Na(+)-dependent succinate transport. ANG I (10(-9) M) and ANG III (10(-10) M) also stimulated the Na(+)-HCO3- cotransporter, and captopril (10(-4) M) attenuated the ANG I stimulation by 68 +/- 3.5% (P < 0.01) but not that of ANG II and III. Saralasin (10(-11) to 10(-8) M) by itself behaved as an agonist, and its stimulation was additive to that by ANG II. The nonpeptide ANG II receptor antagonist, losartan potassium (10(-6) M), and the disulfide-reducing agent, dithiothreitol (10 mM), each by itself had no effect on the cotransporter but each markedly attenuated the ANG II effect (by 77 +/- 1.4%, P < 0.01 and 74 +/- 1.6%, P < 0.005, respectively) in accord with the view that the basolateral receptor belongs to subtype 1. These results identify physiological concentrations of ANG II as a potent, direct, and specific stimulator of the basolateral Na(+)-HCO3- cotransporter.
Assuntos
Angiotensina II/farmacologia , Proteínas de Transporte/metabolismo , Córtex Renal/metabolismo , Angiotensina II/antagonistas & inibidores , Angiotensinas/farmacologia , Animais , Captopril/farmacologia , Cinética , Masculino , Microvilosidades/metabolismo , Concentração Osmolar , Coelhos , Saralasina/farmacologia , Simportadores de Sódio-Bicarbonato , Trocadores de Sódio-HidrogênioRESUMO
Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor identified to date, raising the strong possibility of its involvement in the pathogenesis of systemic hypertension. Whether ET-1 exerts a direct stimulating effect on sodium reabsorption in the renal proximal convoluted tubule, the dominant locus of sodium reabsorption in the nephron, is currently unknown. Such an effect would suggest yet another mechanism by which ET-1 might mediate systemic hypertension. In studies on membrane vesicles prepared from rabbit renal cortex, we show that ET-1 (10(-8) to 10(-11) M) exerts dose-dependent stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter; preincubation of vesicles with 10(-10) M ET-1 for five minutes enhanced the activity of each transporter by approximately 25%. This stimulation reflected an increase in the Vmax of each transporter but no change in the Km for sodium. The stimulatory effect of ET-1 was blocked in the presence of an ET-1 antiserum. Moreover, the stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter by ET-1 displayed specificity as indicated by the lack of effects on the activities of the apical Na(+)-glucose transporter and the basolateral Na(+)-succinate transporter. The data implicate ET-1 as a novel, direct and specific modulator of sodium reabsorption in the proximal tubule. As such, ET-1 might be a direct determinant of extracellular fluid volume under normal and pathophysiologic circumstances, including hypertensive disorders.
Assuntos
Proteínas de Transporte/metabolismo , Endotelinas/farmacologia , Córtex Renal/efeitos dos fármacos , Animais , Bicarbonatos/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Endotelinas/fisiologia , Espaço Extracelular/fisiologia , Técnicas In Vitro , Córtex Renal/metabolismo , Cinética , Masculino , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Coelhos , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato , Trocadores de Sódio-HidrogênioRESUMO
The vacuolar H+ ATPase is inhibited by N-ethylmaleimide (NEM), a sulfhydryl compound, suggesting the involvement of a sulfhydryl group in this transport process. We have examined the effects of several sulfhydryl-containing compounds on the vacuolar H+. ATPase of rabbit renal cortical endosomes. A number of such compounds were effective inhibitors of endosomal H+ transport at 10(-5)-10(-6) M, including NEM, mersalyl, aldrithiol, 5,5' dithiobis (2-nitrobenzoic acid), p-chloromercuribenzoic acid (PCMB) and p-chloromercuriphenyl sulfonic acid (PCMBS). NEM, mersalyl, aldrithiol and PCMBS had no effect on pH-gradient dissipation, whereas PCMB decreased the pH gradient faster than control. In the absence of ATP, PCMB (10(-4) M) stimulated endosomal 36Cl- uptake, particularly in the presence of an inside-alkaline pH gradient (pHin = 7.6/pHout = 5.5). This result was not an effect of PCMB on the Cl(-)-conductive pathway. The less permeable PCMBS did not stimulate 36Cl- uptake. The effects of PCMB were concentration dependent and were prevented by dithioerithritol. ATP-dependent 36Cl- uptake was decreased by addition of PCMB. Finally, PCMB had no effect on 45Ca2+ uptake. These results support the presence of two functionally important sulfhydryl groups in this endosomal preparation. One such group is involved with ATP-driven H+ transport and must be located on the cytoplasmic surface of the endosomal membrane. The second sulfhydryl group must reside on the internal surface of the endosomal membrane and relates to a PCMB-activated Cl-/OH- exchanger that is functional both in the presence and absence of ATP. This endosomal transporter is similar to the PCMB-activated Cl-/OH- exchanger recently described in rabbit renal brush-border membranes.
Assuntos
Cloretos/metabolismo , Córtex Renal/efeitos dos fármacos , ATPases Translocadoras de Prótons/efeitos dos fármacos , Compostos de Sulfidrila/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Técnicas In Vitro , Córtex Renal/enzimologia , Córtex Renal/metabolismo , Cinética , Organelas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Prótons , CoelhosRESUMO
Previous studies have demonstrated a Na(+)-dependent decrease in the ATP-generated acidification of endosomes and have attributed it to the presence of either a Na(+)-H+ exchanger or a Na(+)-K(+)-adenosinetriphosphatase (ATPase) in parallel with the vacuolar H(+)-ATPase. In the present study we have examined the possibility that both of these two Na+ transporters might be present in endosome-enriched microsomes isolated from rabbit renal cortex. After the establishment of a stable pH gradient by ATP in this preparation, addition of Na+ induced a decrease in the pH gradient. Expression of this effect of Na+ did not require the presence of ATP or K+. Choline and K+ had no effect on the ATP-dependent pH gradient, but addition of Li+ caused a small reduction in the pH gradient. Amiloride, ouabain, and vanadate had no effect on the Na(+)-induced dissipation of the ATP-driven pH gradient. In addition, a pH gradient-dependent 22Na+ uptake by the endosomal vesicles that was insensitive to amiloride, ouabain, or vanadate was demonstrated. These results provide evidence against the presence of a Na(+)-K(+)-ATPase in endosome-enriched microsomes from the renal cortex and support the existence of an amiloride-insensitive Na(+)-H+ exchanger in parallel with the vacuolar H(+)-ATPase. This endosomal Na(+)-H+ exchanger might have important implications for the regulation of vacuolar H(+)-ATPase activity as well as proximal tubule acidification.
Assuntos
Proteínas de Transporte/metabolismo , Córtex Renal/metabolismo , Microssomos/metabolismo , Organelas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/farmacologia , Amilorida/farmacologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Córtex Renal/ultraestrutura , Ouabaína/farmacologia , Potássio/farmacologia , Coelhos , Sódio/farmacologia , Trocadores de Sódio-Hidrogênio , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Vanadatos/farmacologiaRESUMO
Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.
Assuntos
Adenosina Trifosfatases/metabolismo , Fosfatase Alcalina/metabolismo , Rim/enzimologia , Microvilosidades/enzimologia , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Transporte Biológico , Cães , Ativação Enzimática , Concentração de Íons de Hidrogênio , Rim/ultraestrutura , Especificidade por Substrato , Fosfolipases Tipo C/metabolismo , Zinco/farmacologiaRESUMO
We have examined the effect of second messengers on ATP-driven H+ transport in an H+ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1 mM) had no effect on H+ transport. Acridine orange fluorescence in the presence of 0.5 mM Ca2+ (+1 mM EGTA) was 19 +/- 6% of control. Inhibition of ATP-driven H+ transport by Ca2+ was concentration dependent; 0.25 and 0.5 mM Ca2+ (+1 mM EGTA) inhibited acridine orange fluorescence by approximately 50 and approximately 80%, respectively. Ca2+ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca2+ did not affect ATP hydrolysis. ATP-dependent Br- uptake was virtually unchanged in the presence of 0.5 mM Ca2+ (+1 mM EGTA). These vesicles were also shown to transport Ca2+ in an ATP-dependent mode. Inositol 1,4,5-trisphosphate had no effect on ATP-dependent Ca2+ uptake. These results are consistent with the co-existence of an H+ ATPase and an H+/Ca2+ exchanger on these endosomes, the latter transport system using the H+ gradient to energize Ca2+ uptake. Attempts to demonstrate an H+/Ca2+ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca2+ uptake could be demonstrated. A Ca2(+)-dependent increase in H+ permeability and an ATP-dependent Ca2+ uptake might have important implications for the regulation of vacuolar H+ ATPase activity as well as the homeostasis of cytosolic Ca2+ concentration.
Assuntos
Adenosina Trifosfatases/metabolismo , Cálcio/farmacologia , Hidrogênio/farmacocinética , Córtex Renal/metabolismo , Animais , Transporte Biológico , Cálcio/farmacocinética , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Guanosina Trifosfato/farmacologia , Hidrogênio/antagonistas & inibidores , Inositol 1,4,5-Trifosfato/farmacologia , Córtex Renal/efeitos dos fármacos , Córtex Renal/enzimologia , Coelhos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
We have examined the activity and kinetic characteristics of the Na+-H+ exchanger in renal cortical brush-border membrane vesicles (BBMV) prepared from rabbits adapted to chronic hypocapnia in order to address whether this transporter might contribute to the suppressed proximal bicarbonate reabsorption characteristic of this disorder. Chronic hypocapnia was induced by exposing animals to 9% O2 for a 5-day period. In comparison with paired, contemporaneous controls, an average delta PaCO2 of 13 mmHg and an average delta [HCO3-] of 7.3 meq/l were obtained. Chronic hypocapnia led to a significant suppression of the 22Na+ uptake by BBMV; at the 3-s mark, a 30% suppression was observed (chronic hypocapnia, 4.05 +/- 0.43 nmol/mg protein; control, 5.72 +/- 0.39 nmol/mg protein) (P less than 0.01). A significant decrease in the Vmax of the antiporter was noted (chronic hypocapnia, 622.7 +/- 86.8 nmol.mg protein-1.min-1; control 857.5 +/- 64.8 nmol.mg protein-1.min-1) (P less than 0.01), whereas the Km for sodium remained unaltered. The specificity of this adaptation was supported by showing that Na+-dependent uptake of D-[3H]glucose by BBMV was not significantly different between chronic hypocapnia and control. Chronic normocapnic hypoxemia left Na+-H+ exchange activity undisturbed. We conclude that the observed change in the BBMV Na+-H+ antiporter might be responsible, at least in part, for the suppressed renal bicarbonate reabsorption characteristic of chronic hypocapnia and that a consequence of the hypocapnic state itself mediates this adaptation.
Assuntos
Dióxido de Carbono/farmacologia , Proteínas de Transporte/metabolismo , Córtex Renal/metabolismo , Aclimatação , Equilíbrio Ácido-Base , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Córtex Renal/efeitos dos fármacos , Cinética , Masculino , Proteínas de Transporte de Monossacarídeos/metabolismo , Coelhos , Valores de Referência , Sódio/metabolismo , Trocadores de Sódio-HidrogênioRESUMO
Two methods are reported for renal membrane preparation from the dog kidney cortex. One method is a simultaneous preparation of brush-border (BBMV) and basolateral (BLMV) membranes. Using readily available laboratory equipment, differential centrifugation produced a supernatant which was treated with Mg2+. The Mg2+ treatment produced a pellet (crude BLMV) which was added to Percoll and centrifuged to produce purified BLMV. The supernatant after Mg2+ treatment eventually yielded pure BBMV after additional Mg2+ precipitations. The second method used an acidic medium in conjunction with divalent-cation precipitation to prepare BBMV. Whichever method was used, BBMV and BLMV showed appropriate enzyme and transport activities.
Assuntos
Membrana Celular/ultraestrutura , Córtex Renal/ultraestrutura , Microvilosidades/ultraestrutura , Animais , Transporte Biológico , Cátions Bivalentes , Fracionamento Celular/métodos , Membrana Celular/metabolismo , Cães , Enzimas/análise , Córtex Renal/metabolismo , Microvilosidades/metabolismo , Ultracentrifugação/métodosRESUMO
An endosomal fraction isolated from rabbit renal cortex by a novel, fast, and simple procedure was enriched in ATP-dependent H+ pumping that was oligomycin insensitive but was inhibited by dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide (NEM), Zn2+, Hg2+, diethylstilbestrol, mersalyl, and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. No substantial Na+-H+ exchange was detected. Electrogenicity of the pump was demonstrated using [14C]-SCN-. In addition, these membranes featured ATP-dependent Cl- flux. The ATP-driven H+ pumping had an absolute requirement for Cl-: an inside-negative membrane potential was not a substitute for Cl-. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-driven Cl- uptake but no inhibition was observed with nigericin. Finally, both ATP-driven H+ pumping and ATP-dependent Cl- flux were inhibited by Cl(-)-channel inhibitors. Part, or all, of the absolute dependence on Cl- may derive from a Cl- channel, the function of which is intimately related to H+ pumping by the ATPase. Flux through this Cl- channel may be regulated by one or more factors, including ATP, membrane potential, and pH.
Assuntos
Trifosfato de Adenosina/farmacologia , Cloretos/farmacologia , Córtex Renal/metabolismo , Organelas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Prótons , Animais , Transporte Biológico Ativo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Membrana Celular/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Diuréticos/farmacologia , Etilmaleimida/farmacologia , Canais Iônicos/metabolismo , Córtex Renal/efeitos dos fármacos , Córtex Renal/ultraestrutura , Nigericina/farmacologia , Oligomicinas/farmacologia , Coelhos , Salicilanilidas/farmacologia , Sódio/metabolismo , Espectrometria de Fluorescência , Tiocianatos/metabolismoRESUMO
The uptake of D-glucose by renal brush border membrane vesicles was studied in the absence of Na+. Uptake of the sugar was membrane potential dependent (inside negative), inhibited by phlorizin, sugar and stereospecific, accelerated by exchange diffusion, saturable, and temperature dependent. The binding of phlorizin in the absence of Na+ was also increased by a membrane potential (inside negative). Thus, the properties of this membrane potential-dependent, Na+-independent sugar transport system resembled those described for the Na+-D-glucose cotransport system. In the absence of Na+ but in the presence of a valinomycin-induced K+ diffusion potential the apparent Km for D-glucose was 43 mM. This contrasted with an apparent Km of 1.8 mM for the Na+ chemical gradient system. Therefore, the Na+-independent uptake system represented a low-affinity transport mechanism. It is suggested that the same carrier mediated the Na+-independent and Na+-dependent transport systems. A hypothetical model for the membrane potential-dependent stimulation of D-glucose uptake in the absence of Na+ is proposed.
Assuntos
Membrana Celular/metabolismo , Glucose/metabolismo , Rim/fisiologia , Microvilosidades/metabolismo , Sódio/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Florizina/farmacologia , Coelhos , Estereoisomerismo , Relação Estrutura-Atividade , Valinomicina/farmacologiaAssuntos
Membrana Celular/metabolismo , Glucose/metabolismo , Túbulos Renais Proximais/metabolismo , Microvilosidades/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Glucose/farmacologia , Gramicidina/farmacologia , Cinética , Lítio/farmacologia , Masculino , Microvilosidades/efeitos dos fármacos , Nistatina/farmacologia , Florizina/farmacologia , Coelhos , Valinomicina/farmacologiaAssuntos
Membrana Celular/metabolismo , Glicerol , Rim/metabolismo , Microvilosidades/metabolismo , Preservação de Tecido , Animais , Transporte Biológico , Glucose/metabolismo , Técnicas In Vitro , Rim/ultraestrutura , Membranas/metabolismo , Concentração Osmolar , Prolina/metabolismo , Coelhos , Temperatura , Preservação de Tecido/métodosAssuntos
Adenosina Trifosfatases/metabolismo , Fosfatidilcolinas/fisiologia , Potássio/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico Ativo , Cação (Peixe) , Ativação Enzimática , Ouabaína/farmacologia , Fosfatidilcolinas/farmacologia , Fosfolipídeos/análise , Potássio/farmacologia , Reto , Glândula de Sal/metabolismo , Sódio/farmacologiaRESUMO
Vesicles containing a purified shark rectal gland (sodium + potassium)-activated adenosine triphosphatase-(NaK ATPase) were prepared by dialyzing for 2 days egg lecithin, cholate, and the NaK ATPase purified from the rectal gland of Squalus acanthias. These vesicles were capable of both Na+ and K+ transport. Studies of K+ transport were made by measuring the ATP-stimulated transport outward of 42K+ or 86Rb+. Vesicles were preloaded with isotope by equilibration at 4 degrees for 1 to 3 days. Transport of 42K+ or 86Rb+ was initiated by addition of MgATP to the vesicles. The ATP-dependent exit of either isotope was the same. Experiments are presented which show that this loss of isotope was not due to changes in ion binding but rather due to a loss in the amount of ion trapped in the vesicular volume. The transport of K+ was dependent on external Mg2+. CTP was almost as effective as ATP in stimulating K+ transport, while UTP was relatively ineffective. These effects of nucleotides parallel their effects on Na+ accumulation and their effectiveness as substrates for the enzyme. Potassium transport was inhibited by ouabain and required the presence of Na+. The following asymmetries were seen: (a) addition of external Mg2+ supported K+ transport; (b) ouabain inhibited K+ transport only if it was present inside the vesicles; (c) addition of external Na+ to the vesicles stimulated K+ transport. External Li+ was ineffective as a Na+ substitute. The specific requirement of external Na+ for K+ transport indicates that K+ exit is coupled to Na+ entry. Changes in the internal vesicular ion concentrations were studied with vesicles prepared in 20 mM NaCl and 50 mM KCl. After 1 hour of transport at 25 degrees, a typical Na+ concentration in the vesicles in the presence of ATP was 72 mM. A typical K+ concentration in the vesicles was 10 mM as measured with 42K+ or 6 mM as measured with 86Rb+. The following relationships have been calculated for Na+ transport, K+ transport and ATP hydrolysis: Na+/ATP = 1.42, K+/ATP =1.04, and Na+/K+ = 1.43. The ratio of 2.8 Na+ transported in to 2 K+ transported out is very close to the value reported for the red cell membrane. Potassium-potassium exchange similar to that observed in the red cell membrane and attributed to the Na+-K+ pump (stimulated by ATP and orthophosphate and inhibited by ouabain) was observed when vesicles were prepared in the absence of Na+. The results reported in this paper prove that the shark rectal gland NaK ATPase, which is 90 to 95% pure, is the isolated pump for the coupled transports of Na+ and K+.